U.S. patent number 10,280,204 [Application Number 15/046,903] was granted by the patent office on 2019-05-07 for electroactive biopolymer optical and electro-optical devices and method of manufacturing the same.
This patent grant is currently assigned to Tufts University. The grantee listed for this patent is TUFTS UNIVERSITY. Invention is credited to Mark Cronin-Golomb, David L. Kaplan, Brian Lawrence, Fiorenzo Omenetto.
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United States Patent |
10,280,204 |
Kaplan , et al. |
May 7, 2019 |
Electroactive biopolymer optical and electro-optical devices and
method of manufacturing the same
Abstract
A method of manufacturing a biopolymer optical device includes
providing a polymer, providing a substrate, casting the polymer on
the substrate, and enzymatically polymerizing an organic compound
to generate a conducting polymer between the provided polymer and
the substrate. The polymer may be a biopolymer such as silk and may
be modified using organic compounds such as tyrosines to provide a
molecular-level interface between the provided bulk biopolymer of
the biopolymer optical device and a substrate or other conducting
layer via a tyrosine-enzyme polymerization. The enzymatically
polymerizing may include catalyzing the organic compound with
peroxidase enzyme reactions. The result is a carbon-carbon
conjugated backbone that provides polymeric "wires" for use in
polymer and biopolymer optical devices. An all organic biopolymer
electroactive material is thereby provided that provides optical
functions and features.
Inventors: |
Kaplan; David L. (Concord,
MA), Omenetto; Fiorenzo (Lexington, MA), Lawrence;
Brian (Minneapolis, MN), Cronin-Golomb; Mark (Reading,
MA) |
Applicant: |
Name |
City |
State |
Country |
Type |
TUFTS UNIVERSITY |
Medford |
MA |
US |
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|
Assignee: |
Tufts University (Medford,
MA)
|
Family
ID: |
39815147 |
Appl.
No.: |
15/046,903 |
Filed: |
February 18, 2016 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20160376331 A1 |
Dec 29, 2016 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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13940168 |
Jul 11, 2013 |
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12513392 |
Nov 5, 2013 |
8574461 |
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PCT/US2007/083639 |
Nov 5, 2007 |
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60856297 |
Nov 3, 2006 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N
33/5436 (20130101); B29D 11/00663 (20130101); G02B
1/04 (20130101); G01N 33/54386 (20130101); G01N
33/544 (20130101); B29D 11/0074 (20130101); C07K
14/43586 (20130101); G01N 33/54373 (20130101); G02B
1/06 (20130101); B29C 39/02 (20130101); G02B
1/046 (20130101); D01F 4/02 (20130101); G02B
6/138 (20130101); B82Y 20/00 (20130101); G02B
2006/12171 (20130101); G02B 2006/12104 (20130101); G02B
2006/1213 (20130101); G02B 5/18 (20130101); G02B
2006/12102 (20130101); G02B 1/045 (20130101); D10B
2211/04 (20130101); D10B 2401/20 (20130101); G02B
1/041 (20130101); G02B 2006/12107 (20130101); G02B
1/005 (20130101); G01N 2333/43578 (20130101) |
Current International
Class: |
C07K
14/435 (20060101); D01F 4/02 (20060101); B29C
39/02 (20060101); B29D 11/00 (20060101); G01N
33/543 (20060101); G02B 1/04 (20060101); G02B
6/138 (20060101); G02B 1/06 (20060101); G01N
33/544 (20060101); G02B 6/12 (20060101); B82Y
20/00 (20110101); G02B 1/00 (20060101); G02B
5/18 (20060101) |
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Primary Examiner: Ahvazi; Bijan
Attorney, Agent or Firm: Quarles & Brady LLP
Government Interests
GOVERNMENT SUPPORT
The invention was made with government support under grant number
EB002520 awarded by the National Institutes of Health, DMR0402489
awarded by the National Science Foundation, and FA9550-04-1-0363
awarded by the United States Air Force. The government has certain
rights in the invention.
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a Continuation of U.S. patent application Ser.
No. 13/940,168, filed on Jul. 11, 2013, which is a Continuation of
U.S. patent application Ser. No. 12/513,392, filed on May 4, 2009,
which is now U.S. Pat. No. 8,574,461, which is a National Stage
Entry of PCT/US2007/083639, entitled "ELECTROACTIVE BIOPOLYMER
OPTICAL AND ELECTRO-OPTICAL DEVICES AND METHOD OF MANUFACTURING THE
SAME" filed Nov. 5, 2007, which claims the benefit of priority of
U.S. Provisional Application Ser. No. 60/856,297 filed on Nov. 3,
2006, entitled "Biopolymer Devices and Methods for Manufacturing
the Same."
Claims
The claimed invention is:
1. An electroactive biopolymer optical device, comprising: a silk
fibroin assembly, comprising: silk fibroin characterized by
beta-sheet secondary structure, wherein the silk fibroin is
genetically altered or chemically modified, wherein the silk
fibroin comprises at least one tyrosine block; at least one
polymeric wire, comprising crosslinked aromatic organic compounds
coupled with the silk fibroin and characterized in that they are
conducting, wherein the crosslinked aromatic organic compounds are
arranged such that form a conducting backbone of the least one
polymeric wire; wherein the at least one polymeric wire is formed
internally within the silk fibroin assembly and/or on a surface of
the silk fibroin assembly; and at least one shaped optical feature,
wherein the at least one shaped optical feature is formed on the
surface of the silk fibroin assembly and/or the at least one shaped
optical feature is or forms a shape of the silk fibroin
assembly.
2. The device of claim 1, wherein the silk fibroin comprises
tyrosine monomers and the crosslinked aromatic organic compounds
comprise tyrosine crosslinking.
3. The device of claim 2, wherein the tyrosine crosslinking
comprises carbon-carbon bonds that form a conducting conjugated
backbone.
4. The device of claim 2, wherein the tyrosine crosslinks form
conjugated conduits along the surface and/or internally within the
silk fibroin assembly.
5. The device of claim 1, wherein the crosslinks form internal
conductive wires or conductive wires on a surface of the
biopolymer.
6. The device of claim 1, wherein the at least one shaped optical
feature is a patterned structure.
7. The device of claim 6, wherein the patterned structure is a
nanopatterned structure.
8. The device of claim 1, wherein the device is selected from the
group consisting of an electro-optical collector, a solar
collector, a mechanical actuator with optical readout, a conformal
coating, and a functionally protective coating.
9. The device of claim 1, wherein the at least one shaped optical
feature is a diffractive or refractive structure selected from the
group consisting of a beam diffuser, a diffraction grating, a
photonic crystal, an optofluidic, a waveguide, a lens, a microlens
array, a nanolens array, a pattern generator, and a beam
reshaper.
10. The device of claim 1, wherein the silk fibroin assembly is a
nanolayer, a nanofiber, or a film.
11. The device of claims 1, further comprising one or more small
organic molecules.
12. The device of claims 1, further comprising organic indicators,
cells, organisms, markers, or proteins.
13. The device of claims 1, further comprising red blood cells,
horseradish peroxidase, phenolsulfonphthalein, nucleic acids, a
dye, a cell, an antibody, enzymes, restriction endonucleases,
ribonucleases, DNA polymerases, glucose oxidase, laccase, tissues,
cells, antibodies, viruses, proteins, peptides, small molecules,
drugs, dyes, amino acids, vitamins, antioxidants, DNA, RNA, RNAi,
lipids, nucleotides, aptamers, carbohydrates, chromophores, light
emitting organic compounds, luciferin, carotenes, light emitting
inorganic compounds, antibiotics, antifungals, antivirals, light
harvesting compounds, chlorophyll, bacteriorhodopsin,
protorhodopsin, porphyrins, or electronically active compounds.
14. The device of claim 1, further comprising a substrate.
15. The device of claims 14, wherein the polymeric wires formed on
the surface of the silk fibroin between the substrate and silk
fibroin.
Description
BACKGROUND OF THE INVENTION
Field of the Invention
The present invention is directed to electroactive biopolymer
optical and electro-optical devices and methods for manufacturing
such devices. The present invention is further directed to
electronics and electrical integration to biopolymer optical and
electro-optical devices.
Description of Related Art
The field of optics is well established. Some subfields of optics
include diffractive optics, micro-optics, photonics and guided wave
optics. Various optical devices have been fabricated in these and
other subfields of optics for research and commercial application.
For example, common optical devices include diffraction gratings,
photonic crystals, optofluidic devices, waveguides, lenses,
microlens arrays, pattern generators, beam reshapers, and the
like.
These optical devices are fabricated using various methods
depending on the application and the optical characteristics
desired. However, these optical devices, and the fabrication
methods employed in their manufacture, generally involve
significant use of non-biodegradable materials. For example, glass,
fused silica, and plastic are commonly used in optical devices.
Such materials are not biodegradable and remain in the environment
for extended periods of time after the optical devices are removed
from service and discarded. Of course, some of the materials can be
recycled and reused. However, recycling also requires expenditures
of natural resources and adds to the environmental costs associated
with such materials.
Therefore, there exists an unfulfilled need for optical devices
that minimize the negative impact to the environment. In addition,
there exists an unfulfilled need for optical devices that provide
additional functional features that are not provided by
conventional optical devices.
SUMMARY OF THE INVENTION
In view of the foregoing, objects of the present invention are to
provide various novel biopolymer optical devices and methods for
manufacturing such optical devices that may be used in various
applications.
One aspect of the present invention is to provide electroactive
biopolymer optical and electro-optical devices.
Another aspect of the present invention is to provide a method for
manufacturing such biopolymer optical devices.
One advantage of the present invention is in providing biopolymer
optical devices that minimize the negative impact to the
environment.
Another advantage of the present invention is in providing
biopolymer optical devices that are biocompatible.
Yet another advantage of the present invention is in providing
biopolymer optical devices that have additional functional features
that are not provided by conventional optical devices.
In the above regard, inventors of the present invention recognized
that biopolymers, and especially silk proteins, present novel
structure and resulting functions. For example, from a materials
science perspective, silks spun by spiders and silkworms represent
the strongest and toughest natural fibers known and present various
opportunities for functionalization, processing, and
biocompatibility. Over five millennia of history accompany the
journey of silk from a sought-after textile to a scientifically
attractive fiber. As much as its features captivated people in the
past, silk commands considerable attention in this day and age
because of its strength, elasticity, and biochemical properties.
The novel material features of silks have recently been extended
due to insights into self-assembly and the role of water in
assembly. These insights, in turn, have led to new processing
methods to generate hydrogels, ultrathin films, thick films,
conformal coatings, three-dimensional porous matrices, solid
blocks, nanoscale diameter fibers, and large diameter fibers.
Silk-based materials achieve their impressive mechanical properties
with natural physical crosslinks of thermodynamically stable
protein secondary structures also known as beta sheets
(.beta.-sheets). As such, no exogenous crosslinking reactions or
post process crosslinking is required to stabilize the materials.
The presence of diverse amino acid side chain chemistries on silk
protein chains facilitates coupling chemistry for functionalizing
silks, such as with cytokines, morphogens, and cell binding
domains. There are no known synthetic or biologically-derived
polymer systems that offer this range of material properties or
biological interfaces, when considering mechanical profiles,
aqueous processing, room-temperature processing, ease of
functionalization, diverse modes of processing, self-forming
crosslinks, biocompatibility, and biodegradability.
Another unique feature provided by the biopolymer devices in
accordance with the present invention, and especially the silk
proteins, is the ability to genetically alter the native sequence
of the biopolymer to add new functions, or to chemically modify the
biopolymer to add new functions. The method and biopolymer devices
of the present invention extend the capabilities of added cell
binding domains (RGD), redox triggers (methionines for
oxidation/reduction control), and phosphorylation triggers
(enzymatic kinase/phosphatase reactions). The biopolymer optical
devices of the present invention further genetically redesign new
versions of silk that retain native silk assembly and structure,
but offer additional functions.
Additionally, using methods of the present invention, a variety of
aromatic organic compounds, including tyrosines, can be
enzymatically polymerized to generate conducting polymers. The
polymerization of the organic compounds may be performed from
solutions or in the solid state. This enzymatic process may be
catalyzed by peroxidase enzyme reactions and is based on free
radical coupling. The result is a carbon-carbon conjugated backbone
that provides polymeric "wires" for use in polymer and biopolymer
optical devices.
Further modifications to biopolymers such as silk may be made with
tyrosines, either genetically or via chemical coupling. Tyrosines
provide a molecular-level interface between the bulk silk protein
and the optical features with a conducting layer or features via
tyrosine-enzyme polymerization. Correspondingly, a unique,
all-organic biopolymer electroactive material may be realized that
also provides optical features.
More specifically, in accordance with the present invention,
tyrosine monomers can be enzymatically crosslinked to form
conducting polymers. The optical gratings made of biopolymers such
as silk may be re-engineered to genetically encode tyrosine blocks
in the silk. Tyrosine crosslinking may be used to form conducting
wires, and further controls may be implemented to control the
position at which the "wires" are formed, both internally and on
the surface of the silk. As such, the present invention provides
directed integration of electronic components into the biopolymer
optical devices. These biopolymer materials, including silk, can be
used for electronic properties for new conformal coatings and
related technologies and include additional optical features.
The tyrosine moieties may be incorporated in the polymer or
biopolymer such as silk via genetic engineering or via surface
chemistry as a "functional" fusion component. For example,
carbodiimide coupling may be used to incorporate the tyrosine
moieties. Subsequent post processing polymerization via enzymatic
processes generates conjugated conduits along the silk protein
assemblies. The polymerization step is based on a secondary
enzymatic polymerization with peroxidase to stitch the tyrosine
carbon to carbon (C to C) bonds together to generate conducting
polymers. The ability to form nanolayers, nanofibers, and related
material systems with precise control of conducting polymer
location and features provides new options for forming conformal,
light weight, functional protective coatings with enhanced
electronic and optical functions for a variety of applications.
Peroxidase catalysis, mediated by hydrogen peroxide, was used to
form conducting polymers from a wide range of aromatic compounds.
Horseradish peroxidase (HRP) is a glycoprotein that contains a
single-chain .beta.-type hemoprotein with an Fe containing
porphyrin. HRP catalysis of aromatic compounds was used to form the
conducting polymers. The solid-state polymerization reactions of
aromatics on surfaces, via peroxidase catalyzed reactions, was used
to form conducting polymers. In one embodiment of the present
invention, dip-pen nanolithography (DPN) was used to pattern
4-aminothiophenol and tyrosines as the "ink". DPN patterning of an
aromatic monomer, with surface induced orientation, was used to
promote enzymatic polymerization under ambient surface reactions to
form conducting polymers.
An example surface reaction may include a 0.01 M H.sub.2O.sub.2
stock solution, prepared by diluting H.sub.2O.sub.2 water solution
(30% w/w) with MeOH/H.sub.2O (1:1 by volume) mixture. Peroxidase or
hematin catalyzed polymerization may be carried out by immersing
the solid state assemblies (either self-standing or on the surface
of glass slides) into the H.sub.2O.sub.2 stock solution, which
contains 200 .mu.L horseradish peroxidase stock solution. The silk
assembly is washed by dipping it in buffer solutions several times
after the reaction. The peroxidase (donor: hydrogen peroxide
oxidoreductase; EC 1.11.1.7, Type II, from horseradish, and hematin
(procine) are commercially available.
Hematin provides benefits in the solid state material reactions due
to the smaller size of the molecule compared to horseradish
peroxidase, which relates to diffusion of the tyrosines not at the
surface of the bulk materials, for example, with internal blocks. A
typical hematin reaction includes sodium phosphate buffer, the silk
material, and hematin. An equal molar amount of hydrogen peroxide
(0.6 mmol) is added as oxidant, as in the peroxidase reactions.
Various applications exist for the electroactive biopolymer devices
in accordance with the present invention. For example, the
electroactive biopolymer devices may be used as electro-optical
collectors, solar collectors, mechanical actuators with optical
readout, and in other applications where light-weight, degradable,
electroactive devices are desired.
While no other biopolymer or synthetic polymer can match the range
of features outlined above for silk, the inventors of the present
invention have identified some other polymers that exhibit various
properties similar or analogous to silk. In particular, other
natural biopolymers including chitosan, collagen, gelatin, agarose,
chitin, polyhydroxyalkanoates, pullan, starch (amylase
amylopectin), cellulose, hyaluronic acid, and related polymers have
been identified. In view of the above-noted features of biopolymers
and of silk in particular, the present invention provides novel
biopolymer optical devices and methods for manufacturing such
devices.
In accordance with one aspect of the present invention, a method of
manufacturing a conducting polymer includes providing a polymer,
providing a substrate, casting the polymer on the substrate, and
enzymatically polymerizing an organic compound to generate a
conducting polymer between the provided polymer and the substrate.
In one embodiment, the polymer provided is a biopolymer, such as
silk, while in other embodiments, other polymers may be used
including chitosan, collagen, gelatin, agarose, chitin,
polyhydroxyalkanoates, pullan, starch (amylose amylopectin),
cellulose, hyaluronic acid, polydimethylsiloxane (PDMS), and
related biopolymers, or variations and combinations thereof.
In one embodiment, the polymer may be a matrix solution, and the
polymerization is performed from the matrix solution, while in
other embodiments, the polymer may be a solid, and the
polymerization is performed from the solid. Additionally, in one
embodiment, the enzymatic polymerization may include catalyzing the
organic compound with peroxidase enzyme reactions. For example, in
one embodiment, tyrosine is used as the organic compound, while in
other embodiments, other organic compounds may be used including
red blood cells, horseradish peroxidase, phenolsulfonphthalein,
nucleic acid, a dye, a cell, an antibody, enzymes, for example,
peroxidase, lipase, amylose, organophosphate dehydrogenase,
ligases, restriction endonucleases, ribonucleases, DNA polymerases,
glucose oxidase, laccase, cells, viruses, proteins, peptides, small
molecules (e.g., drugs, dyes, amino acids, vitamins, antioxidants),
DNA, RNA, RNAi, lipids, nucleotides, aptamers, carbohydrates,
chromophores, light emitting organic compounds such as luciferin,
carotenes and light emitting inorganic compounds (such as chemical
dyes), antibiotics, antifungals, antivirals, light harvesting
compounds such as chlorophyll, bacteriorhodopsin, protorhodopsin,
and porphyrins and related electronically active compounds, or
variations and combinations thereof.
In one embodiment of the present invention, an interface is formed
between the polymer and the substrate, where the interface is a
conducting layer formed by tyrosine-enzyme polymerization. The
interface may include a carbon to carbon (C to C) conjugated
backbone.
In one embodiment, the substrate may act as a mold or a template
for an optical device. The substrate may be a mold or template for
electro-optical collectors, solar collectors, mechanical actuators
with optical readout, and other applications where light-weight,
degradable, electroactive devices are desired. The substrate may
also be a mold of template for biopolymer devices including lenses,
microlens arrays, optical gratings, pattern generators, beam
reshapers, and the like. Other geometric features, such as holes
and pits, for example, may also be included in the substrate.
In accordance with another embodiment of the present invention, a
conducting polymer is provided that is made of at least a bulk
protein, a substrate, an organic compound, and an enzyme that
polymerizes the organic compound to generate a conducting polymer
between the polymer and the substrate. The conducting polymer may
be a biopolymer, such as silk, or may be another polymer including
chitosan, collagen, gelatin, agarose, polyhydroxyalkanoates,
pullan, starch (amylose amylopectin), cellulose, hyaluronic acid,
polydimethylsiloxane (PDMS), and related biopolymers, or
combinations thereof.
These and other advantages and features of the present invention
will become more apparent from the following detailed description
of the preferred embodiments of the present invention when viewed
in conjunction with the accompanying drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1A and 1B are schematic flow diagrams illustrating a method
of manufacturing a biopolymer optical device in accordance with one
embodiment of the present invention.
FIG. 2 is a graph that illustrates the relationship between the
volume of 8% silk concentration vs. film thickness.
FIG. 3A is a photograph of a biopolymer film made of silk.
FIG. 3B is a graph showing the prism coupled angular dependence of
reflectivity of the biopolymer film of FIG. 8A.
FIG. 3C is a graph showing the measured transmission of light
through the biopolymer film of FIG. 8A.
FIG. 4 illustrates results graphs showing retention of the
hemoglobin function within an RBC-doped silk optical device.
FIG. 5 is a photograph showing diffractive biopolymer devices that
have been cast in silk, chitosan, and collagen.
FIG. 6 is a graph showing antibody IgG1 activity related to initial
activity in the silk films prepared in the two different formats
and stored at the three different temperatures.
FIG. 7 is a graph showing antibody IgG activity related to initial
activity in the silk films prepared in the two different formats
and stored at the three different temperatures.
DETAILED DESCRIPTION OF THE INVENTION
In view of the superior functional characteristics and
processability noted above, the biopolymer optical devices in
accordance with the present invention are described as being
fabricated using a biopolymer such as silk. In this regard, the
silk utilized was silkworm silk. However, there are many different
silks, including spider silk, transgenic silks, and genetically
engineered silks, variants and combinations thereof and others,
that may alternatively be used to manufacture biopolymer optical
devices in accordance with the present invention.
In addition, other biodegradable polymers may be used instead of
silk. For example, other biopolymers, such as chitosan, exhibit
desirable mechanical properties, can be processed in water, and
form generally clear films for optical applications. Other
biopolymers, such as collagen, cellulose, chitin, hyaluronic acid,
amylase, and the like may alternatively be utilized in specific
applications. Synthetic biodegradable polymers such as polyactic
acid, polyglycolic acid, polyhydroxyalkanoates, and related
copolymers may also be selectively used. Such polymers may be used
by themselves, or in combination with silk and other polymers and
may be used to manufacture biopolymer optical devices for specific
applications.
FIG. 1A is a schematic illustration of a flow diagram 10 showing a
method of manufacturing a biopolymer optical device in accordance
with one embodiment of the present invention. If a biopolymer is
provided in step 11, the process proceeds to step 16 below.
Otherwise, a biopolymer is provided in step 12. In the example
where the biopolymer is silk, the biopolymer may be provided by
extracting sericin from the cocoons of Bombyx muni. In one
embodiment, the biopolymer may be a solution such as a biopolymer
matrix solution, while in other embodiments, different solvents
other than water, or a combination of water and other solvents may
be used, depending on the biopolymer used.
In the example of silk, an aqueous silk fibroin solution may be
processed, for example, 8.0 wt %, which is then used to manufacture
the biopolymer optical device. Of course, in other embodiments, the
solution concentrations may also be varied from very dilute
(approximately 1 wt %) to very high (up to 30 wt %) using either
dilution or concentration, for example, via osmotic stress or
drying techniques. In this regard, other embodiments may utilize
different percent weight solutions to optimize flexibility or
strength of the resultant nanopatterned biopolymer optical device,
depending on the application. Production of aqueous silk fibroin
solution is described in detail in WIPO Publication Number WO
2005/012606 entitled "Concentrated Aqueous Silk Fibroin Solution
and Uses Thereof," which is incorporated by reference.
Additionally, the polymer may be a solid, and the polymerization is
then performed using the solid.
A substrate is provided in step 16 to serve as a mold or template
in manufacturing the biopolymer optical device. A surface of the
substrate has the desired characteristic features to be formed on
the biopolymer optical device. In this regard, the substrate may be
an appropriate nanopattern on a surface of the optical device and
may be an optical device such as a nanopatterned optical grating or
other optical device, depending on the optical features desired for
the device being manufactured. The polymer, such as the aqueous
biopolymer matrix solution or the solid described above, is cast on
the substrate in step 18. Upon drying, and upon completion of the
subsequent reactions, a solidified biopolymer film is formed on the
surface of the substrate. The thickness of the biopolymer film
depends on the volume of the biopolymer matrix solution or the
solid polymer applied to the substrate.
Patterned nanostructures can be provided on the biopolymer films,
such as the silk films manufactured in the above discussed manner.
In one embodiment, the surface of the substrate may be smooth so as
to provide a smooth biopolymer film, and a nanopattern may be
machined on the surface of the biopolymer film. The nanopattern may
be machined using a laser, such as a femtosecond laser, or by other
nanopattern machining techniques, including lithography techniques
such as photolithography, electron beam lithography, and the like.
Using such techniques, nanopattern features as small as 700 nm that
are spaced less than 3 .mu.m have been demonstrated as described in
further detail below.
In another embodiment, the surface of the substrate itself may have
an appropriate nanopattern thereon so that when the solidified
biopolymer film is removed from the substrate, the biopolymer film
is already formed with the desired nanopattern on a surface
thereof. In such an implementation, the substrate may be an optical
device such as a nanopatterned optical grating, depending on the
nanopattern desired on the biopolymer films. The substrate surfaces
may be coated with Teflon.TM. and other suitable coatings to ensure
even detachment after the biopolymer matrix solution transitions
from the liquid to the solid phase. The ability of the biopolymer
casting method using a nanopatterned substrate for forming highly
defined nanopatterned structures in the resultant biopolymer films
was verified, and silk films having nanostructures as small as 75
nm and RMS surface roughness of less than 5 nm have been
demonstrated.
Referring again to FIG. 1A, in step 20, an organic compound is
enzymatically polymerized to generate a conducting polymer between
the provided polymer and the substrate. The enzymatic reaction
genetically alters the native sequence of the silk protein to add
new functions or chemically modifies the biopolymer to add new
functions, depending upon the polymer (for example, the silk
protein) chosen and the enzyme reaction components. The method of
the present invention extends the capabilities of added cell
binding domains (Arginine-Glycine-Aspartic acid--RGD), redox
triggers (methionines for oxidation/reduction control), and
phosphorylation triggers (enzymatic kinase/phosphatase reactions).
The enzymatic polymerization of the silk proteins of the present
invention further genetically redesigns new versions of silk that
retain native silk assembly and structure, but offer additional
functions.
In one embodiment, the polymerized organic compound may be aromatic
organic compounds such as amino acids, including tyrosines, that
can be enzymatically polymerized to generate conducting polymers.
As indicated above, the polymerization of the organic compounds may
be performed from a solution or from a solid state.
Further modifications to biopolymers such as silk may be made with
tyrosines, either genetically or via chemical coupling. Tyrosines
provide a molecular-level interface between the bulk silk protein
and the optical features with a conducting layer or with features
via tyrosine-enzyme polymerization. Correspondingly, a unique,
all-organic biopolymer electroactive material may be realized that
also provides optical features.
More specifically, as shown in step 210, tyrosine monomers can be
enzymatically crosslinked to form conducting polymers. The optical
gratings made of biopolymers such as silk may be re-engineered to
genetically encode tyrosine blocks in the silk as shown in step
212.
As shown in step 22 in FIG. 1B, the enzymatic process may be
catalyzed by peroxidase enzyme reactions and is based on free
radical coupling. For example, peroxidase catalysis, mediated by
hydrogen peroxide, was used to form conducting polymers from a wide
range of aromatic compounds. Horseradish peroxidase (HRP) is a
glycoprotein that contains a single-chain .beta.-type hemoprotein
with an Fe containing porphyrin. HRP catalysis of aromatic
compounds was used to form the conducting polymers. The solid-state
polymerization reactions of aromatics on surfaces, via peroxidase
catalyzed reactions, was used to form conducting polymers. In one
embodiment of the present invention, dip-pen nanolithography (DPN)
was used to pattern 4-aminothiophenol and tyrosines as the "ink".
DPN patterning of an aromatic monomer, with surface induced
orientation, was used to promote enzymatic polymerization under
ambient surface reactions to form conducting polymers.
The tyrosine moieties can be incorporated in the biopolymer such as
silk via genetic engineering or via surface chemistry as a
"functional" fusion component. For example, carbodiimide coupling
may be used to incorporate the tyrosine moieties. Subsequent post
processing polymerization via enzymatic processes generates
conjugated conduits along the silk protein assemblies. As shown in
step 214, the polymerization step is based on a secondary enzymatic
polymerization with peroxidase to stitch the tyrosine carbon to
carbon (C to C) bonds together to generate conducting polymers. The
ability to form nanolayers, nanofibers, and related material
systems with precise control of conducting polymer location and
features provides new options for forming conformal, light weight,
functional protective coatings with enhanced electronic and optical
functions for a variety of applications.
An example surface reaction may include a 0.01 M H.sub.2O.sub.2
stock solution, prepared by diluting H.sub.2O.sub.2 water solution
(30% w/w) with MeOH/H.sub.2O (1:1 by volume) mixture. Peroxidase or
hematin catalyzed polymerization can be carried out by immersing
the solid state assemblies (either self-standing or on the surface
of glass slides) into the H.sub.2O.sub.2 stock solution which
contains 200 .mu.L horseradish peroxidase stock solution. The silk
assembly is washed by dipping it in buffer solutions several times
after the reaction. The peroxidase (donor: hydrogen peroxide
oxidoreductase; EC 1.11.1.7), Type II, from horseradish, and
hematin (procine) are commercially available.
Hematin provides benefits in the solid state material reactions due
to the smaller size of the molecule compared to horseradish
peroxidase, which may relate to diffusion of the tyrosines not at
the surface of the bulk materials, for example, with internal
blocks. A typical hematin reaction includes sodium phosphate
buffer, the silk material, and hematin. An equal molar amount of
hydrogen peroxide (0.6 mmol) would be added as oxidant, as in the
peroxidase reactions.
As shown in step 24, tyrosine crosslinking may be used to form
conducting polymeric "wires" for the biopolymer optical device,
resulting from a carbon-carbon (C to C) conjugated backbone. As
shown in step 36, further controls may be implemented to control
the position at which the "wires" are formed, both internally and
on the surface of the silk. As such, directed integration of
electronic components into the biopolymer optical devices may be
performed in accordance with the present invention. These
biopolymer materials, such as silk, can be used for electronic
properties for new conformal coatings and related technologies and
include additional optical features.
Various applications of the electroactive biopolymer devices
include use as electro-optical collectors, solar gelatin,
mechanical actuators with optical readout, and other applications
where light-weight, degradable, electroactive devices can be
used.
Experiments were conducted to validate the above-described method
by manufacturing various biopolymer optical waveguides. The
relationship between the volume of 8 wt % silk concentration
aqueous silk fibroin solution, and the resulting silk film
thickness, is shown in the graph 30 of FIG. 2, where the aqueous
silk fibroin solution was cast over a substrate surface of
approximately 10 square centimeters. The X-axis shows the volume of
silk fibroin solution in mL, and the Y-axis shows the thickness of
the resultant film in .mu.m.
Of course, the film properties such as thickness and biopolymer
content, as well as optical features, may be altered based on the
concentration of fibroin used in the process, the volume of the
aqueous silk fibroin solution or solid deposited, and the
post-deposition process for drying the cast solution to lock in the
structure. Accurate control of these parameters is desirable to
ensure the optical quality of the resultant biopolymer optical
waveguide and to maintain various characteristics of the biopolymer
optical waveguide, such as transparency, structural rigidity, and
flexibility. Furthermore, additives to the biopolymer matrix
solution may be used to alter features of the biopolymer optical
waveguide such as morphology, stability, and the like, as known
with polyethylene glycols, collagens, and the like.
An unpatterned biopolymer film having a thickness of 10 .mu.m was
manufactured in the above-described manner using an aqueous silk
fibroin solution, and was characterized in a scanning prism coupled
reflectometer from Metricon Corporation. FIG. 3A illustrates the
unpatterned biopolymer film 34 manufactured and characterized. The
index of refraction of the biopolymer film 34 was measured to be
n=1.55 at 633 nm, which is slightly higher than the index of
refraction of conventional borosilicate glass. The measured index
of refraction confirms that the value is high enough to afford
reasonable contrast for optical use such as in air-silk biophotonic
crystals (BPC) (.DELTA.n.sub.fibroin-.DELTA.n.sub.air=0.55). The
characterization of the unpatterned silk film 34 is shown in graph
36 of FIG. 3B, which clearly demonstrates the prism coupled angular
dependence of the reflectivity. The oscillations in graph 36 are
due to coupling into guided waves, demonstrating the use of silk as
a waveguide material.
The measured roughness of cast silk film on an optically flat
surface shows measured root mean squared roughness values between
2.5 and 5 nanometers, which implies a surface roughness easily less
than .lamda./50 at a wavelength of 633 nm. Atomic force microscope
images of patterned silk diffractive optics show the levels of
microfabrication obtainable by casting and lifting silk films off
of appropriate molds. The images show definition in the hundreds of
nanometer range and the sharpness of the corners indicates the
possibility of faithful patterning down to the tens of
nanometers.
In addition, the unpatterned silk film 34 was also analyzed to
determine transparency. FIG. 3C is a graph 38 that illustrates the
measured transmission of light through the silk film 34 in various
wavelengths. Transmission measurements indicate that the
unpatterned silk film 34 was highly transparent across the visible
spectrum. For comparison, similar thickness films were also cast in
collagen, and polydimethylsiloxane (PDMS). The free-standing
structural stability was found to be inferior, and the resultant
biopolymer optical device was not self-supporting when implemented
as a thin film. However, such biopolymers may be used in other
applications if structural stability is deemed to be not as
important.
Importantly, shaped films having various thicknesses were patterned
on the nanoscale using the method of FIG. 1 described above to
provide nanopatterned biopolymer optical devices.
The term "nanopatterned" as used with regard to the present
invention refers to very small patterning that is provided on a
surface of the biopolymer optical device. The patterning has
structural features whose size can be appropriately measured on a
nanometer scale (that is, 10.sup.-9 meters), for example, sizes
ranging from 100 nm to few microns. Additionally, the biopolymer
optical devices of the present invention may incorporate various
different optical devices such as lenses, diffraction gratings,
photonic crystals, waveguides, and the like.
A variety of nanopatterned biopolymer optical devices were
successfully manufactured using the above-described method of the
present invention using silk fibroin solution. These devices
included waveguides, lenses, microlens arrays, optical gratings,
pattern generators, and beam reshapers. In particular, the aqueous
solution of silk fibroin was cast onto specific substrates with
patterns thereon. The substrate surfaces were coated with
Teflon.TM. to ensure even detachment after the biopolymer matrix
solution transitions from the liquid to the solid phase. The
ability of the biopolymer casting method of the present invention
for forming highly defined nanopatterned structures in biopolymer
optical devices was verified by casting the optical waveguides of
the present invention. Regular patterned features with dimensions
down to 210 nm, and localized surface roughness of less than 20 nm,
have been attained. As mentioned above, smoothing techniques may
also be used to further reduce or remove surface roughness of the
biopolymer optical waveguide.
Such regular patterning of biocompatible materials allows
manufacturing of optical devices that can be used to provide
photonic bandgaps and manipulate light via an organic, yet
mechanically robust optical device. These devices combine the
flexibility of embedded optics with the unique versatility of the
protein substrate as explained throughout the application. Many
advantages are provided by the present invention including
combining the organic nature of biopolymers such as silk with the
power of diffractive and transmissive optics embedded in an organic
matrix to create biologically active optical elements. Silk
provides a controllably degradable, biocompatible, and structurally
strong medium with which to fabricate the optical devices in
accordance with the present invention.
Transmissive nanopatterned diffractive biopolymer optical devices
were made using the method of the present invention described
above. These optical devices include biopolymer optical waveguides,
silk diffusers, line pattern generators, and cross pattern
generators. Such optical devices use appropriately configured
wavelength scale surface structuring to create predefined one or
two-dimensional light patterns that exploit light interference.
Such optical devices made of conventional materials have been
applied to imaging, spectroscopy, beam sampling and transformation,
and metrology to name a few uses. Extending this approach to
control the delivery of light within a biological matrix such as
silk biopolymer can provide optimal coupling of photons into a
substrate or allow for designed optical discrimination, interface,
or readout.
A significant advantage of biopolymer optical waveguides in
accordance with the present invention is the ability of the optical
waveguides to be biologically activated since they are entirely
organic and biocompatible. Water-based processing can be used, for
example, for silk optical waveguides. This increases cellular
survivability of the waveguides and the likelihood of
biocompatibility.
To confirm biocompatibility of nanopatterned biopolymer optical
devices, red blood cells (RBCs) were incorporated into a silk
diffraction grating in accordance with the present invention that
was manufactured as described above with regard to FIG. 1. The
RBC-silk fibroin solution was prepared by combining 1 ml of an 80%
hematocrit human RBC solution and 5 ml of the 8% silk solution. The
mixture was cast on a 600 lines/mm optical grating and allowed to
dry overnight. The film was removed from the optical grating and
annealed for two hours. The grating structure was observed in the
resultant RBC-doped silk diffraction grating.
The RBC-doped silk diffraction grating was then tested to observe
the diffraction orders. An optical transmission experiment was
performed to determine whether hemoglobin (the oxygen-carrying
protein contained in RBCs) maintained its activity within the
matrix of the silk diffraction grating. The results graphs 160 are
shown in FIG. 4 and indicate the retention of hemoglobin function
within the RBC-doped silk diffraction grating. The X-axis
corresponds to the wavelength (in nm), and the Y-axis indicates the
absorbance by the RBC-doped silk diffraction grating.
In particular, the RBC-doped silk diffraction grating was inserted
in a quartz cuvette filled with distilled water, and an absorbance
curve was observed. This result is shown by line (b) HbO.sub.2 in
results graphs 160. As can be seen, the absorbance curve shown by
line (b) HbO.sub.2 exhibited two peaks typical of oxy-hemoglobin
absorption. Subsequently, nitrogen gas was bubbled into the cuvette
to deoxygenate the hemoglobin. After 15 minutes, the characteristic
absorption peaks of oxy-hemoglobin disappeared from the absorbance
curve. This result is shown by line (a) Hb in the results graphs
160. These results were further confirmed when the nitrogen flow to
the cuvette is subsequently halted, which resulted in the
reappearance of the oxy-hemoglobin peaks. This result is shown by
line (c) HbO.sub.2 in results graphs 160.
As previously noted, alternative biopolymers may also be used for
fabrication of nanopatterned biopolymer optical devices in
accordance with the present invention. FIG. 5 shows a photograph
180 that illustrates other diffractive biopolymer optical devices
that have been cast using different materials. In particular, a
chitosan optical device 182 and a collagen optical device 184 have
also been manufactured in accordance with the present invention.
With respect to chitosan, optical diffraction characteristics
similar to silk have been observed.
It should be evident from the above discussion and the example
nanopatterned biopolymer optical devices shown and discussed that
the present invention provides biodegradable biopolymer optical
devices. High quality biopolymer optical devices were manufactured
that are naturally biocompatible, can be processed in water, and
can undergo degradation with controlled lifetimes. As explained
above, the biopolymer optical devices of the present invention may
also be biologically activated by incorporating small organic
materials. In particular, the biopolymer optical devices can be
biologically functionalized by optionally embedding it with one or
more organic indicators, living cells, organisms, markers,
proteins, and the like. More specifically, the biopolymer optical
devices in accordance with the present invention may be embedded or
coated with organic materials such as red blood cells, horseradish
peroxidase, phenolsulfonphthalein, nucleic acid, a dye, a cell, an
antibody, as described further in Appendix I, enzymes, for example,
peroxidase, lipase, amylose, organophosphate dehydrogenase,
ligases, restriction endonucleases, ribonucleases, DNA polymerases,
glucose oxidase, laccase, cells, viruses, proteins, peptides, small
molecules (e.g., drugs, dyes, amino acids, vitamins, antioxidants),
DNA, RNA, RNAi, lipids, nucleotides, aptamers, carbohydrates,
chromophores, light emitting organic compounds such as luciferin,
carotenes and light emitting inorganic compounds (such as chemical
dyes), antibiotics, antifungals, antivirals, light harvesting
compounds such as chlorophyll, bacteriorhodopsin, protorhodopsin,
and porphyrins and related electronically active compounds, tissues
or other living materials, other compounds or combinations thereof.
The embedded organic materials are biologically active, thereby
adding biological functionality to the resultant biopolymer optical
device.
The embedding of the biopolymer optical devices with organic
materials may be performed for example, by adding such materials to
the biopolymer matrix solution used to manufacture the biopolymer
films, such as the silk fibroin matrix solution. In the
implementation where the biopolymer optical device is manufactured
using a solid, the optical device can be biologically
functionalized by functionalizing of one or more of the polymer
films.
The present invention broadens the versatility of optical devices
by allowing the direct incorporation of labile biological receptors
in the form of peptides, enzymes, cells, antibodies, or related
systems, and the like and allows such optical devices to function
as biological sensing devices.
The biopolymer optical devices of the present invention can be
readily used in environmental and life sciences where
biocompatibility and biodegradability are paramount. For example,
the nanopatterned biopolymer optical devices as described above can
be unobtrusively used to monitor a natural environment such as in
the human body and may be implanted in vivo without a need to
retrieve the device at a later time. The degradation lifetime of
the biopolymer optical devices of the present invention can be
controlled during the manufacturing process, for example, by
controlling the ratio and amount of the solution matrix cast or the
type of polymer used. Moreover, the biopolymer optical devices of
the present invention can be dispersed in the environment, again
without the need to retrieve them at a later time, thereby
providing novel and useful devices for sensing and detection.
The foregoing description of the aspects and embodiments of the
present invention provides illustration and description, but is not
intended to be exhaustive or to limit the invention to the precise
form disclosed. Those of skill in the art will recognize certain
modifications, permutations, additions, and combinations of those
embodiments are possible in light of the above teachings or may be
acquired from practice of the invention. Therefore the present
invention also covers various modifications and equivalent
arrangements that fall within the purview of the appended
claims.
Antibody Stability in Silk Films
Materials--Anti-IL-8 monoclonal antibody (IgG1) was purchased from
eBioscience, Inc. human polyclonal antibody IgG and human IgG ELISA
Quantitation Kit were purchased from Bethyl Laboratories Inc. All
other chemicals used in the study were purchased from Sigma-Aldrich
(St. Louis, Mo.).
Antibody entrapment in silk films--human polyclonal antibody
IgG--Ten ml 1 mg/ml IgG mixed with 167 ml 6% silk solution make the
IgG concentration in silk film mg/g silk. 100 .mu.l of mixed IgG
solution was added to each well of 96 well plate which was placed
in a fume hood with cover opened overnight. The dried film was
either treated or not treated with methanol. For methanol
treatment, the wells were immersed in 90% methanol solution for 5
min and dried in the fume hood. All dry 96 well plates were then
stored at 4.degree. C., room temperature, and 37.degree. C. (FIG.
6).
Anti-IL-8 monoclonal antibody (IgG1)--0.5 ml 1 mg/ml IgG1 mixed
with 83 ml 6% silk solution make the IgG1 concentration in silk
film 0.1 mg/g silk. 50 .mu.l of mixed IgG1 solution was added to a
well of 96 well plate which was placed in a fume hood with cover
opened overnight. The dried film was either treated or not treated
with methanol. For methanol treatment, the wells were immersed in
90% methanol solution for 5 min and dried in the fume hood. All dry
96 well plates were then stored at 4.degree. C., room temperature,
and 37.degree. C. (FIG. 7).
Antibody measurement--Five wells prepared at the same condition
were measured for statistic. Pure silk (without antibody) was used
as a control.
For non methanol-treated samples, 100 .mu.l of PBS buffer, pH 7.4,
was added to the well which was further incubated at room
temperature for 30 min to allow the film to completely dissolve.
Aliquot of solution was then subjected to antibody measurement. For
methanol-treated samples, 100 .mu.l HFIP was added into each well
which was further incubated at room temperature for 2 hours to
allow the film completely dissolve. The silk HFIP solution was
dried in a fume hood overnight. The follow step was the same as non
methanol-treated samples, added PBS buffer and pipette the solution
for antibody measurement.
ELISA--Polystyrene (96-well) microtitre plate was coated with 100
.mu.L of antigen anti-Human IgG-affinity at a concentration of 10
.mu.g/mL prepared in antigen coating buffer (bicarbonate buffer, 50
mM, pH 9.6) and then incubated overnight storage at room
temperature. The wells were then washed three times with TBS-T
buffer. The unoccupied sites were blocked with 1% BSA in TBS (200
.mu.L each well) followed by incubation for 30 minutes at room
temperature. The wells were then washed three times with TBS-T. The
test and control wells were then diluted with 100 .mu.L of serially
diluted serum. Each dilution was in TBS buffer. Serially diluted
blanks corresponding to each dilution were also present. The plate
was then incubated for 1 h at room temperature. The plate was
washed again with TBS-T buffer (five times). Bound antibodies were
assayed with an appropriate conjugate of anti-human IgG-HRP
(1:100,000), 100 .mu.L of it was coated in each well and kept at
room temperature for 1 hour. Washing of the plate with TBS-T (five
times) was followed by addition of 100 .mu.L TMB in each well and
incubation at room temperature for 5-20 min. The absorbance of each
well was monitored at 450 nm on a VersaMax microplate reader
(Molecular devices, Sunnyvale, Calif.).
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